PDGF-B基因沉默对LPS介导小鼠乳腺上皮细胞分泌IL-6和TNF-α的影响

Effects of PDGF-B gene silencing on secretion of IL-6 and TNF-α in LPS-stimulated mammary epithelial cell

旨在观察PDGF—B基因沉默对LPS诱导小鼠乳腺上皮细胞分泌IL-6和TNF—α的影响，为揭示乳腺上皮细胞的免疫机制提供参考数据。采用siRNA转染技术对PDGF—B基因进行沉默，用实时荧光定量RT—PCR方法筛选最佳转染条件，并检测沉默后LPS介导其下游信号P13KmRNA的表达量，用ELISA法检测其下游信号P13K蛋白含量和LPS介导细胞分泌前炎症细胞因子IL-6和TNF—α的含量。结果显示，选用PDGF-B-mus-886片段的50nmol／L浓度转染组能极显著抑制PDGF-B的mRNA表达（P〈0．01）；PDGF—B沉默后，LPS刺激下其下游P13K的mRNA和蛋白表达量与对照组相比均呈显著性降低（P〈0．05）；细胞分泌IL-6的量与对照组相比呈显著性降低（P〈0．05），而分泌TNF-α的量各组无显著性变化（P〉0．05）。结果表明，PDGF-B可引起LPS介导乳腺上皮细胞IL-6分泌量的改变，因此，PDGF-B可作为靶基因深入研究乳腺上皮细胞的免疫功能，为乳腺炎症、肿瘤等相关乳腺疾病的治疗提供新的思路及试验数据，为新药的开发和应用提供理论依据。

This experiment was conducted to study the effect of PDGF-B on secretion of IL-6 and TNF-α in LPS-stimulated mammary epithelial cells （MECs）, so as to reveal the immune mecha- nism of MECs. PDGF-B gene was transfected with RNA interference, then the transfection effi- ciency was detected by real-time quantitative RT-PCR. Meanwhile,the PI3K mRNA was detected with the same method. The content of PI3K protein, IL-6 and TNF-α in the mouse MECs were measured by ELISA. The results showed that,PDGF-B-mus-886 in 50 nmol/L could significantly inhibited PDGF-B mRNA expression（P〈0.01）. When the mouse MECs was transfected with siR- NA of PDGF-B and stumilated with LPS,compared with the control group,both the expression of PI3K mRNA and protein were reduced significantly after the silence of PDGF-B gene （P〈0.05）. The secretion of IL-6 was prominently reduced （P〈0.05） ,but there was no change （P〉0.05） in the TNF-α content in each group. These results indicated that the silence of PDGF-B could change the expression of IL-6 in LPS-stimulated MECs, so PDGF-B could be chosen as a target gene to further explore the immune function of mouse MECs. It provides a new train of thought and exper- imental data for treatment of mammary gland inflammation, tumor and other related mammary gland disease, and lays a theoretical basis for the development and application of new drugs.